Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disorder characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3 per million population per year in the Western world, but 3 times higher in East Asia. Survival in severe aplastic anemia (SAA) has improved significantly due to advances in hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy, biologic agents, and supportive care. In SAA, HSCT from a matched sibling donor (MSD) is the first-line treatment. If a MSD is not available, options include immunosuppressive therapy (IST), matched unrelated donor, or haploidentical HSCT. The purpose of this guideline is to provide health care professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary evidence-based document prepared by a group of pediatric hematologists of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Hemato-Oncology (AIEOP) was discussed, modified and approved during a series of consensus conferences that started online during COVID 19 and continued in the following years, according to procedures previously validated by the AIEOP Board of Directors.

FIP1L1-PDGFRA in chronic eosinophilic leukemia and BCR-ABL1 in chronic myeloid leukemia affect different leukemic cells.

We investigated genetically affected leukemic cells in FIP1L1-PDGFRA+ chronic eosinophilic leukemia (CEL) and in BCR-ABL1+ chronic myeloid leukemia (CML), two myeloproliferative disorders responsive to imatinib. Fluorescence in situ hybridization specific for BCR-ABL1 and for FIP1L1-PDGFRA was combined with cytomorphology or with lineage-restricted monoclonal antibodies and applied in CML and CEL, respectively. In CEL the amount of FIP1L1-PDGFRA+ cells among CD34+ and CD133+ cells, B and T lymphocytes, and megakaryocytes were within normal ranges. Positivity was found in eosinophils, granulo-monocytes and varying percentages of erythrocytes. In vitro assays with imatinib showed reduced survival of peripheral blood mononuclear cells but no reduction in colony-forming unit growth medium (CFU-GM) growth. In CML the BCR-ABL1 fusion gene was detected in CD34+/CD133+ cells, granulo-monocytes, eosinophils, erythrocytes, megakaryocytes and B-lymphocytes. Growth of both peripheral blood mononuclear cells and CFU-GM was inhibited by imatinib. This study provided evidence for marked differences in the leukemic masses which are targeted by imatinib in CEL or CML, as harboring FIP1L1-PDGFRA or BCR-ABL1.

Genomic gain at 6p21: a new cryptic molecular rearrangement in secondary myelodysplastic syndrome and acute myeloid leukemia.

Fluorescence in situ hybridization and comparative genomic hybridization characterized 6p rearrangements in eight primary and in 10 secondary myeloid disorders (including one patient with Fanconi anemia) and found different molecular lesions in each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different breakpoints along the 6p arm. Reciprocal translocations were found in seven. In the 10 patients with secondary acute myeloid leukemia/myelodysplastic syndrome (AML/MDS), the short arm of chromosome 6 was involved in unbalanced translocations in 7. The other three patients showed full or partial trisomy of the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two patients). In 5/7 patients with unbalanced translocations, DNA sequences were overrepresented at band 6p21 as either cryptic duplications (three patients) or cryptic low-copy gains (two patients). In the eight patients with cytogenetic or cryptic 6p gains, we identified a common overrepresented region extending for 5-6 megabases from the TNF gene to the ETV-7 gene. 6p abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p gains are major pathogenetic events arising from acquired and/or congenital genomic instability.

Incidence and significance of cryptic chromosome aberrations detected by fluorescence in situ hybridization in acute myeloid leukemia with normal karyotype.

To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: -5, -7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P < 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53deletion or trisomy 8 (one case each) in 33-60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76-94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.

Cytogenetic and FISH investigations on tetrasomy 8 in ANLL.

Two patients with "de novo" ANLL and tetrasomy of chromosome 8 at diagnosis are described. A mosaic karyotype with coexistence of normal metaphases was found in both cases. Trisomy 8 was also present in one metaphase of the first patient. Fluorescent in situ hybridization with a centromeric probe from chromosome 8 was applied in the second case, confirming the presence of a minor population with trisomy 8 in interphase nuclei.

Interphase FISH for Y chromosome, VNTR polymorphisms, and RT-PCR for BCR-ABL in the monitoring of HLA-matched and mismatched transplants.

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.

Identification of chromosome changes in acute myeloid leukemia (AML-M2) by molecular cytogenetics.

Karyotyping with fluorescence in situ hybridization (FISH) is reported in two rare cases of AML-M2 FAB. In the first case FISH analysis confirmed the presence of a t(7;11)(p15:p15) translocation in a complex karyotype that also showed an unbalanced translocation involving the other chromosome 7, a rare rearrangement between chromosomes 9 and 20, and four or five copies of a small marker derived from chromosome 9. In the second case whole chromosome painting with probes for chromosomes 8, 14, and 21 revealed the presence of a masked t(8;21) translocation in which one chromosome 14 was involved in a newly discovered rearrangement, i.e., t(8;14;21)(q22-q24;q11;q22). Moreover , double color FISH using ETO-CDR P1 probe and a cosmid for the 5' part of AML-1 on chromosome 21 showed a two color signal on the 8q-, suggesting a recombination between ETO and AML 1. Molecular cytogenetics overcomes limitation of chromosome banding in the interpretation of complex rearrangements.

Trisomy 6 is the hallmark of a dysplastic clone in bone marrow aplasia.

Clonal hematopoiesis with trisomy 6 as the sole karyotypic change was revealed by cytogenetics in two cases of aplastic anemia. In both patients, dyserythropoiesis was characterized by asynchrony of maturation between nucleus and cytoplasm, binucleated elements, and intercytoplasmic connections. In addition to conventional cytogenetics, the size of the trisomic clone was evaluated by fluorescence in situ hybridization on fixed cells at diagnosis and in the course of the disease by using an alpha-satellite centromeric probe for chromosome 6. Moreover, in situ hybridization on bone marrow smears showed that dysplastic erythrocytes as well as myeloid cells belonged to the trisomic clone. Trisomy 6 identifies a subgroup of hematologic disorders with bone marrow hypo-aplasia and dyserythropoiesis.

Associated vascular lesions in patients undergoing coronary artery bypass grafting.

Atherosclerotic involvement of extracoronary arteries in patients undergoing myocardial revascularization can cause severe postoperative complications and increase postoperative mortality. Between January and November 1998, routine preoperative echo-Doppler study of carotid vessels, abdominal aorta and iliac-femoral arteries was performed in all patients undergoing coronary artery bypass grafting (CABG) at our institution, in order to assess the prevalence and the degree of associated vascular lesions. Correlations between echo-Doppler findings, angiographic patterns of coronary lesions and atherosclerotic risk factors were analyzed in all cases. Among 302 patients undergoing CABG, 186 (61.6%) had carotid disease, with a haemodynamically significant stenosis (>70%) of internal carotid in 31 (10.2%). Twenty-three patients had asymptomatic severe carotid disease. A significant correlation between severity of coronary disease and prevalence of severe carotid disease was found (p = 0.02). An abdominal aortic dilatation (diameter > 25 mm) was found in 20 cases (6.6%), with a diameter >35 mm in 7 patients (2.3%), 6 with triple-vessel coronary disease, and 1 with double-vessel disease. Atherosclerotic lesions of iliac-femoro-popliteal axis were found in 165 (54.6%) patients, with a strong correlation to the severity of coronary disease (p = 0.02); lesions were haemodynamically significant (> 70%) in 48 (15.8%) cases. Symptoms of carotid and peripheral vascular disease are no reliable predictors of perioperative risk in patients undergoing CABG. Non-invasive complete arterial investigation should be routinely performed in these patients, in order to plan the most suitable operative approach and to prevent perioperative vascular complications.

Short-term results of bovine internal mammary artery use in cardiovascular surgery.

Over a 14-month period, 28 bovine internal mammary arteries (Bioflow, Bio-Vascular, Inc.; St. Paul, Minnesota, USA) were implanted in 20 patients at our institutions. In 8 patients, the bovine internal mammary artery was used to bypass coronary vessels: in 4 of these patients, coronary artery bypass grafting was performed because of coronary disease (1 type-I aortic dissection); in the other 4 (all with aortic dissection), the modified Bentall technique was used for coronary artery reimplantation. In the remaining 12 patients, the bovine artery was used in vascular surgery: as a graft for lower-extremity occlusive disease (4 patients), arteriovenous fistula (2 patients), and aorticorenal bypass (1 patient); and as a patch to the carotid bifurcation or the common femoral artery in association with endarterectomy (5 patients). The 21 bovine grafts were all 5 mm in diameter; the 7 bovine patches were 4 mm. Of the 8 coronary bypass patients, 2 who underwent coronary artery bypass grafting had acute postoperative myocardial infarctions, and 2 who underwent the modified Bentall technique died in surgery. Follow-up angiography showed complete bovine internal mammary artery graft occlusion in 2 patients; 2 symptom-free patients refused examination. Of the vascular surgery patients, 1 with mild left leg claudication had graft occlusion, shown by angiography, 4 months after surgery. Renal scintigraphy performed in the patient with aorticorenal bypass 4 months after operation showed no blood flow to the kidney. Two patients died for reasons unrelated to bovine mammary artery grafting. The remaining patients are well and free of complications. In view of the high incidence of early occlusion, we do not recommend use of the bovine internal mammary artery graft in coronary surgery. In vascular surgery, the results are more encouraging; however, studies comprising a larger number of patients and longer follow-up are needed to determine whether the use of the bovine internal mammary artery graft can be recommended.

[Prevalence of vascular disease in candidates for myocardial revascularization with aortocoronary bypass: review of the literature and practical implications]. / Prevalenza di patologia vascolare in soggetti candidati a rivascolarizzazione miocardica mediante bypass aortocoronarico: revisione della letteratura ed implicazioni pratiche.

BACKGROUND: Cardiovascular disease remains the main cause of death and morbidity in the industrialized world. Atherosclerosis is a slowly progressive disease; coronary artery disease may be the first presentation of a systemic pathology. The association between coronary artery disease and peripheral vascular disease has often been confirmed by multicenter trials; nevertheless it still remains a subject of debate. METHODS: In order to assess the incidence of coronary artery disease and the degree of associated vascular lesions, between January 1997 and September 1999, in the Department of Cardiothoracic Surgery of the Second University of Naples (Italy), all candidates to coronary artery bypass grafting (CABG) were submitted to routine preoperative echo color Doppler study of the carotid vessels, abdominal aorta and iliac-femoral arteries. The correlation between the echo color Doppler findings, the angiographic patterns of coronary lesions and atherosclerotic risk factors was analyzed in all cases. RESULTS: Among 540 patients undergoing CABG, 418 (77.4%) had carotid disease, with a stenosis > 70% in 62 (11.3%). Forty-nine (79%) patients had asymptomatic severe carotid disease. A significant correlation between the severity of coronary disease and the incidence of severe carotid disease was found (p = 0.02). An abdominal aortic dilation was found in 37 cases (6.7%). Its diameter exceeded 35 mm in 14 patients (2.5%) and in 8 it was associated with triple vessel coronary disease. Atherosclerotic lesions of the iliac-femoro-popliteal axis were found in 394 (72.9%) patients and strongly correlated with the severity of coronary artery disease (p = 0.02); lesions were hemodynamically significant in 91 (16.8%) cases. CONCLUSIONS: Our study emphasizes the association between coronary artery disease and vascular disease. Non-invasive complete arterial investigation should be routinely performed in patients undergoing CABG.